SH2B1 Defends Against Energy Imbalance, Obesity, and Metabolic Disease via a Paraventricular Hypothalamus→Dorsal Raphe Nucleus Neurocircuit

Abstract SH2B1 mutations are associated with obesity, type 2 diabetes, and metabolic dysfunction‐associated steatotic liver disease (MASLD) in humans. Global deletion of Sh2b1 results in severe obesity, type 2 diabetes, and MASLD in mice. Neuron‐specific restoration of SH2B1 rescues the obesity phenotype of Sh2b1‐null mice, indicating that the brain is a main SH2B1 target. However, SH2B1 neurocircuits remain elusive. SH2B1‐expressing neurons in the paraventricular hypothalamus (PVHSH2B1) and a PVHSH2B1→dorsal raphe nucleus (DRN) neurocircuit are identified here. PVHSH2B1 axons monosynaptically innervate DRN neurons. Optogenetic stimulation of PVHSH2B1 axonal fibers in the DRN suppresses food intake. Chronic inhibition of PVHSH2B1 neurons causes obesity. In male and female mice, either embryonic‐onset or adult‐onset deletion of Sh2b1 in PVH neurons causes energy imbalance, obesity, insulin resistance, glucose intolerance, and MASLD. Ablation of Sh2b1 in the DRN‐projecting PVHSH2B1 subpopulation also causes energy imbalance, obesity, and metabolic disorders. Conversely, SH2B1 overexpression in either total or DRN‐projecting PVHSH2B1 neurons protects against diet‐induced obesity. SH2B1 binds to TrkB and enhances brain‐derived neurotrophic factor (BDNF) signaling. Ablation of Sh2b1 in PVHSH2B1 neurons induces BDNF resistance in the PVH, contributing to obesity. In conclusion, these results unveil a previously unrecognized PVHSH2B1→DRN neurocircuit through which SH2B1 defends against obesity by enhancing BDNF/TrkB signaling.


Introduction
Obesity-associated genes predominantly regulate the central nervous system in humans, highlighting the pivotal role of the brain, particularly the hypothalamus, in obesity pathogenesis. [1]The paraventricular hypothalamus (PVH) is long recognized as a satiety center, and ablation of PVH neurons causes hyperphagia and obesity. [2]he PVH encompasses diverse neuronal subpopulations, which express distinct neuropeptides, receptors, and signaling proteins (e.g., oxytocin, prodynorphin, MC4R, GLP-1R, and HTR2c). [3]1b,4] In the acuate nucleus (ARC), POMC neurons and AgRP neurons, which sense circulating nutrient and hormonal cues, directly project to the PVH. [5]In the PVH, MC4R neurons are a target of POMC and AgRP neurons to inhibit food intake. [6]3b,7] SH2B1 is a SH2 domain-containing protein and mediates cell signaling in response to various growth factors, hormones, and cytokines, including growth hormone, insulin, leptin, plateletderived growth factor, nerve growth factor (NGF), and brainderived neurotrophic factor (BDNF). [8]In mice, global deletion of Sh2b1 causes hyperphagia, obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD, previously called NAFLD). [9]8b,10] Importantly, neuron-specific restoration of SH2B1 completely rescues the hyperphagic and obese phenotypes of Sh2b1-null mice. [11]These observations suggest that neuronal SH2B1 is required for defense against energy imbalance and obesity in both humans and rodents.To explore SH2B1 pathways in vivo, we deleted Sh2b1 specifically in leptin receptor (LepRb) expressing neurons.LepRb neuron-specific ablation of Sh2b1 abrogates the ability of leptin to stimulate sympathetic nerve activity, suppresses brown adipose tissue (BAT) thermogenesis, and causes obesity, insulin resistance, and MASLD. [12]Unexpectedly, food intake is normal in the mutant mice; hence, SH2B1 neurons, which regulate appetite, remain unknown.1a,14] These observations raise the possibility that SH2B1 may regulate appetite and food intake by enhancing the BDNF/TrkB pathway in hypothalamic neurocircuits.
The dorsal raphe nucleus (DRN) emerges as an important neural center to control energy balance, body weight, and metabolism. [15]DRN neurons receive inputs from both the forebrain and the hindbrain and project to multiple downstream brain areas. [3e, 15,16] The DRN encompasses serotonergic (DRN 5-HT ), glutamatergic (DRN Vglut3 ), and GABAergic (DRN Vgat ) neurons.5] DRN 5-HT neurons directly activate POMC neurons via 5-HT 2C R in the ARC to reduce food intake.[16i,l,m,17] Stimulation of the DRN 5-HT →ARC circuit inhibits feeding in mice.[16j] DRN 5-HT neurons also project to the ventral tegmental area (VTA), lateral hypothalamus (LH), and bed nucleus of the stria terminalis (BNST).[16j,k] In addition, DRN 5-HT neurons promote adipose thermogenesis and defend against hyperglycemia and hyperlipidemia.[18] Like DRN 5-HT neurons, DRN Vglut3 neurons also suppress appetite and food intake.6b] In this study, we show that SH2B1expressing neurons in the PVH (PVH SH2B1 ) project to the DRN.Optogenetic activation of the PVH SH2B1 →DRN circuit suppresses appetite.Deletion of Sh2b1 in DRN-projecting PVH SH2B1 neurons causes obesity, insulin resistance, and MASLD.SH2B1 counteracts energy imbalance and obesity at least in part by enhancing BDNF action. These results unveil a previously unrecognized PVH SH2B1 →DRN neurocircuit as well as SH2B1-based mechanism shaping circuit activity.

L. Rui Division of Gastroenterology and Hepatology Department of Internal Medicine University of Michigan Medical School
Ann Arbor, MI 48109, USA

Deletion of Sh2b1 in PVH Neurons Causes Obesity
To explore the SH2B1 function in the PVH, we generated SIM1 neuron-specific Sh2b1 knockout mice (Sh2b1 ΔSIM1 ) by crossing Sh2b1 f/f mice with Sim1-Cre mice.Sh2b1 f/f and Sim1-Cre mice were described previously. [6,19]To validate PVH-targeting, Sim1-Cre mice were crossed with Cre-dependent green fluorescent protein (GFP) reporter mice.GFP expression was restricted to the PVH (Figure S1A, Supporting Information).To further confirm PVH-specific deletion of Sh2b1, hypothalamic sections were stained with anti-SH2B1 antibody.SH2B1 immunoreactivity was substantially reduced in the PVH but not in other hypothalamic regions of Sh2b1 ΔSIM1 mice, compared to Sh2b1 f/f mice (Figure S1B, Supporting Information).We monitored the body weights of these mice on a normal chow diet.Remarkably, Sh2b1 ΔSIM1 males and females developed severe obesity (Figure 1A).Whole body fat mass was significantly higher in Sh2b1 ΔSIM1 mice relative to sex-matched Sh2b1 f/f littermates (Figure 1B).Epididymal white adipose tissue (eWAT) and inguinal WAT (iWAT) weights were markedly higher in Sh2b1 ΔSIM1 than in Sh2b1 f/f males (Figure 1C).Adipocyte size in iWAT and eWAT was substantially larger in Sh2b1 ΔSIM1 than in Sh2b1 f/f littermates (Figure 1D).In Sh2b1 ΔSIM1 female mice, gonadal WAT (gWAT) and iWAT weights were also significantly higher and lipid droplets were dramatically larger (Figure 1C; Figure S1C, Supporting Information).Lipid droplets in BAT were larger in Sh2b1 ΔSIM1 mice (Figure 1D; Figure S1C, Supporting Information).These results demonstrate that SIM1 neuron-intrinsic SH2B1 is required for counteracting obesity.

Sh2b1 𝚫SIM1 Mice Develop Glucose Intolerance, Insulin Resistance, and MASLD
We next examined the metabolic function of PVH-intrinsic SH2B1 and performed glucose tolerance test (GTT) and insulin tolerance test (ITT) on Sh2b1 ΔSIM1 and Sh2b1 f/f mice at 15 weeks of age (on a chow diet).Blood glucose levels were significantly higher in Sh2b1 ΔSIM1 males and females relative to sex-matched Sh2b1 f/f mice (Figure 2A; Figure S2A, Supporting Information).Mice were fasted overnight and injected with insulin.Insulin-stimulated phosphorylation of hepatic AKT (pThr308, pSer473) was markedly lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 2B,C).Hepatic lipid droplets were larger and more abundant and liver triacylglycerol (TAG) levels were significantly higher in Sh2b1 ΔSIM1 males and females relative to sex-matched Sh2b1 f/f mice (Figure 2D,E; Figure S2B, Supporting Information).Liver expression of lipogenic genes (Srebp1c, Chrebp, Fasn) and lipid uptake gene (CD36) was higher in Sh2b1 ΔSIM1 mice (Figure 2F).Considering that chronic inflammation promotes metabolic disorders, we accessed cytokine expression.Liver expression of Tnf, Il1, and Mcp1 and adipose expression of Il1 were significantly higher in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 2F,G).Of note, GTT and ITT were comparable between Sh2b1 ΔSIM1 and Sh2b1 f/f mice prior to their body weight divergence (Figure S2C,D, Supporting Information).These data demonstrate that disruption of Sh2b1 in SIM1 neurons results in obesity-associated insulin resistance, glucose intolerance, and MASLD.

Deletion of Sh2b1 in PVH Neurons Induces Energy Imbalance
We sought to interrogate the underlying mechanism of obesity.We examined energy balance by CLAMS in Sh2b1 f/f and Sh2b1 ΔSIM1 littermates (on a chow diet) at 9 weeks of age when their body weights began to diverge (Figure S3A, Supporting Information).Food intake was significantly higher in Sh2b1 ΔSIM1 than in Sh2b1 f/f littermates (Figure 3A).In the dark phase, O 2 consumption and CO 2 production (normalized to lean mass) were considerably lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 3B; Figure S3B, Supporting Information).The respiratory exchange ratio (RER) was significantly lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 3B; Figure S3B, Supporting  C) Mice (20 weeks old) were fasted overnight and injected with insulin.Liver extracts were immunoblotted with the indicated antibodies.AKT phosphorylation was normalized to total AKT levels.Sh2b1 f/f : n = 3, Sh2b1 ΔSIM1 : n = 4. D) H&E and Oil Red O staining of liver sections at 18 weeks of age (three mice per group).Scale bar: 200 μm.E) Liver TAG levels at 20 weeks of age (normalized to liver weight).Sh2b1 f/f : n = 8, Sh2b1 ΔSIM1 : n = 6.F,G) mRNA abundance at 20 weeks of age (normalized to 36B4 levels).Liver Sh2b1 f/f : n = 6, liver Sh2b1 ΔSIM1 : n = 7. iWAT Sh2b1 f/f : n = 6, iWAT Sh2b1 ΔSIM1 : n = 6.a.u.: arbitrary units.All data were from male mice.Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, C,E-G) 2-tailed unpaired Student's t-test or A) two-way ANOVA (A).Information).Analysis of covariance (ANCOVA) further confirmed a decrease in energy expenditure in Sh2b1 ΔSIM1 mice (Figure S3C, Supporting Information).Locomotor activity (dark phase) and body core temperature (both dark and light phases) were significantly lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 3C,D).BAT expression of thermogenic genes (Ucp1, Prdm16, Ppar, Pgc1) was markedly lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice (Figure 3E).UCP1 protein levels were dramatically lower in Sh2b1 ΔSIM1 mice (Figure 3F,G).BAT sections were stained with antibodies to tyrosine hydroxylase (TH), a sympathetic nerve marker.TH + fibers and puncta (cross-sections) were dramatically reduced in Sh2b1 ΔSIM1 mice compared to Sh2b1 f/f littermates (Figure 3H).TH protein levels in BAT extracts were significantly lower in Sh2b1 ΔSIM1 mice (Figure 3F,G).These results suggest that PVH SH2B1 neuron-intrinsic SH2B1 suppresses food intake while increasing energy expenditure.

Adult-Onset Deletion of Sh2b1 in PVH Neurons Causes Obesity and Metabolic Disorders
Cre is also expressed in a few non-PVH areas in Sim1-Cre mice, [6] raising a concern that in Sh2b1 ΔSIM1 mice, Sh2b1 might be also deleted in these non-PVH regions.To address this concern, adult Sh2b1 f/f males (8 weeks old) were bilaterally microinjected into the PVH with adeno-associated virus (AAV1)-hSyn-Cre (deleting Sh2b1 in PVH neurons) or AAV1-hSyn-GFP vector (control) (Figure 4A; Figure S4A, Supporting Information).Body weight diverged between the two groups within one week after AAV injection (Figure 4B).AAV1-hSyn-Cre mice gained over 27 g of body weight within 7 weeks, whereas AAV1-hSyn-GFP mice gained ≈3.3 g.Total fat mass, but not lean mass, was drastically higher in AAV1-hSyn-Cre than in AAV1-hSyn-GFP mice 5 weeks after AAV injection (Figure 4C).In GTT and ITT, blood glucose levels were markedly higher in AAV1-hSyn-Cre than in AAV-hSyn-GFP mice 6 weeks after AAV transduction (Figure 4D).Food intake was significantly higher in AAV1-hSyn-Cre mice (Figure 4E).AAV1-hSyn-Cre mice displayed cold intolerance (Figure S4B, Supporting Information).These results confirm that PVH neuron-specific deletion of Sh2b1 causes hyperphagia, obesity, and metabolic disorders independently of brain development.

PVH SH2B1 Neuron-Specific Overexpression of SH2B1 Protects Against Diet-Induced Obesity
To determine whether overexpression of SH2B1 confers metabolic benefits, we prepared an AAV-CAG-DIO-SH2B1 vector expressing human SH2B1 in a Cre-dependent manner (Figure S4C, Supporting Information).AAV-CAG-DIO-SH2B1 plasmids were cotransfected with or without Cre plasmids into N2a cells, a neuronal line.SH2B1 was detected in the presence, but not in the absence, of Cre (Figure S4D, Supporting Information).To validate the viral vector in vivo, the AAV9-CAG-DIO-SH2B1 vector was co-microinjected into mouse cortices with or without the AAV-hSyn-Cre vector.Cre expression substantially increased cortical SH2B1 levels (Figure S4E, Supporting Information).To deliver SH2B1 specifically into PVH SH2B1 neurons, Sh2b1-IRES-eGFP-2A-Cre (referred to as Sh2b1-Cre) mice were bilaterally microinjected into the PVH with AAV9-CAG-DIO-SH2B1 vector.Wild-type (WT) mice were used as controls.In Sh2b1-Cre mice, an IRES-eGFP-2A-Cre gene cassette was knocked in the Sh2b1 locus (after the STOP codon) to generate GFP and Cre under the control of the endogenous Sh2b1 promoter. [12]Indeed, endogenous SH2B1 and GFP were colocalized in PVH neurons (Figure S4F, Supporting Information).AAV9-CAG-DIO-SH2B1 transduction increased SH2B1 levels in the PVH in Sh2b1-Cre mice relative to WT mice (Figure 4F).To visually confirm PVH SH2B1 neuron-specific targeting, Sh2b1-Cre mice were bilaterally injected into the PVH with a Cre-dependent AAV9-CAG-DIO-mCherry reporter.PVH SH2B1 neurons were marked by mCherry (Figure S4G, Supporting Information).

PVH SH2B1 Neurons Project to the DRN
We set out to map PVH SH2B1 neurocircuits.Sh2b1-Cre males were unilaterally microinjected into the PVH with an AAV9-hSyn-DIO-mCherry vector (Figure 5A).PVH SH2B1 neuronal somas were marked by mCherry as expected (Figure 5B).PVH SH2B1 axonal fibers (mCherry + ) were very abundant in the DRN (Figure 5B).PVH SH2B1 projections to the  periaqueductal gray (PAG) were also detected but to a substantially lower level.To examine monosynaptic connectivity, Sh2b1-Cre males were microinjected into the PVH with a Cre-dependent AAV1-hSyn-DIO-mGFP-2A-Synaptophysin-mRuby vector.Synaptophysin is an integral membrane protein of presynaptic vesicles, and synaptophysin-mRuby protein localizes in nerve terminals to mark synapse formation. [20]We detected abundant synaptophysin-mRuby puncta in the DRN (Figure 5C).These results suggest that PVH SH2B1 neurons form monosynaptic connections with DRN neurons.
We next examined PVH SH2B1 neuronal activation by measuring the expression of c-Fos, a commonly used neuronal activation marker.In wild-type mice, c-Fos + neurons in the PVH were higher in the fed than in the fasted conditions (Figure S5C, Supporting Information).Importantly, PVH c-Fos + neurons were significantly lower in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice under fed conditions (Figure S5D,E, Supporting Information).These results raise the possibility that PVH SH2B1 neuron-intrinsic SH2B1 is involved in central metabolic sensing and integration.

Overexpression of SH2B1 in DRN-Projecting PVH Neurons Protects Against HFD-Induced Obesity
C57BL/6J males were bilaterally microinjected into the PVH with Cre-dependent AAV9-CAG-DIO-SH2B1 vector while the DRN was injected with retrograde AAV-hSyn-Cre or AAV-hSyn-GFP (control) vector (Figure 6G).This paradigm resulted in SH2B1 overexpression specifically in a PVH subpopulation projecting to the DRN.Mice were fed an HFD after one week of recovery.Body weight diverged in 2 weeks after AAV transduction and then became progressively and markedly lower in AAV-hSyn-Cre mice (SH2B1 overexpression) relative to AAV-hSyn-GFP mice (Figure 6H).Total fat mass was significantly lower in AAV-hSyn-Cre mice relative to AAV-hSyn-GFP mice 7 weeks after HFD (Figure 6I).AAV-hSyn-Cre mice displayed significant improvements in glucose intolerance (GTT) and insulin resistance (ITT) compared with AAV-hSyn-GFP mice (Figure 6J).Liver TAG levels were significantly lower in AAV-hSyn-Cre mice relative to AAV-hSyn-GFP mice 11 weeks after AAV transduction (Figure 6K).Liver lipid droplets were smaller and less abundant in AAV-hSyn-Cre mice (Figure S7D, Supporting Information).In light of these findings, we propose that PVH SH2B1 neurons sense/integrate, via SH2B1 pathways, neuronal and hormonal signals and relay metabolic information to the DRN, thereby shaping the ability of DRN neurons to control energy balance, body weight, and metabolism (Figure 6L).

PVH SH2B1 Neuron-Intrinsic SH2B1 Mediates the Anti-Obesity Action of PVH BDNF
To deliver BDNF to the PVH, we generated an AAV-CAG-proBDNF vector (encoding the full length of mouse BDNF).We validated the vector and BDNF expression/secretion in N2a culture as well as in mouse brains (Figure S9A,B, Supporting Information).AAV9-CAG-proBDNF vector was bilaterally microinjected into the PVH of Sh2b1 ΔSIM1 or Sh2b1 f/f males to chronically deliver BDNF to the PVH.We confirmed that BDNF overexpression was restricted to the PVH and was not spread to the VMH (Figure S9A, Supporting Information).Endogenous BDNF was below the detection threshold.BDNF caused weight loss in Sh2b1 f/f but not Sh2b1 ΔSIM1 mice (Figure S9B,C, Supporting Information).Total fat mass, but not lean mass, was significantly higher in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice 5 weeks after BDNF expression (Figure 7G).Glucose intolerance and insulin resistance were more severe in Sh2b1 ΔSIM1 than in Sh2b1 f/f mice after BDNF expression (Figure S9D, Supporting Information).

Discussion
The PVH encompasses heterogeneous neuronal populations projecting to broad brain areas.Distinct PVH neurocircuits play unique and important roles in controlling energy balance, metabolism, and body weight.In this work, we mapped a previously unrecognized PVH SH2B1 →DRN neurocircuit that pivotally controls body weight and metabolism (Figure 6L).The DRN emerges as an important energy balance center. [15]VH SH2B1 neurons sent axons to the DRN and formed monosynaptic connections with DRN neurons.Target neuron phenotypes (e.g., DRN 5-HT , DRN Vglut3 , and/or DRN Vgat ) are currently unknown.Optogenetic stimulation of PVH SH2B1 axonal fibers in the DRN rapidly suppressed feeding behavior.Chronic inhibition of PVH SH2B1 neurons causes obesity, glucose intolerance, insulin resistance, and MASLD.Considering that DRN 5-HT and DRN Vglut3 neurons suppress appetite, we are tempted to propose that PVH SH2B1 neurons directly activate anorexigenic DRN 5-HT and/or DRN Vglut3 neurons, thereby inhibiting food intake.In line with this notion, glutamatergic neurons were reported to be predominant in the PVH. [7,21]Importantly, glutamatergic neurons accounted for a large portion of DRN-projecting PVH SH2B1 neurons.Additionally, PVH SH2B1 neurons may also stimulate DRN 5-HT and/or DRN Vglut3 neurons indirectly through local interneurons in the DRN.
We demonstrated that PVH SH2B1 neuron-intrinsic BDNF/TrkB/SH2B1 pathways safeguard the function of the PVH SH2B1 →DRN neurocircuit.Both embryonic-onset and adultonset deletion of Sh2b1 in PVH neurons caused hyperphagia, reduced energy expenditure, obesity, insulin resistance, glucose intolerance, and MASLD.Likewise, deletion of Sh2b1 in DRN-projecting PVH SH2B1 neurons, using the Cre-dependent, Flp-dependent, and retrograde AAV paradigm, also caused energy imbalance, obesity, insulin resistance, and MASLD.Conversely, overexpression of SH2B1 in either total or DRNprojecting PVH SH2B1 neurons protected against HFD-induced obesity, metabolic disorders, and MASLD.Thus, SH2B1 levels in PVH SH2B1 neurons critically influence body weight, raising the possibility that SH2B1 may act as a molecular rheostat to control energy balance and metabolism.These findings point to a new anti-obesity strategy by upregulating or activating hypothalamic SH2B1.
Given that LepRb-expressing neurons are sparse in the PVH, it is unlikely that SH2B1 in the PVH protects against obesity by directly enhancing leptin signaling.We found that deletion of Sh2b1 in either total or DRN-projecting PVH SH2B1 neurons inhibited BDNF signaling in the PVH.Conversely, overexpression of SH2B1 enhanced PVH BDNF signaling.8g] Remarkably, ablation of Sh2b1 in PVH SH2B1 neurons abrogated the ability of PVH BDNF to protect against hyperphagia, obesity, and metabolic disorders.Collectively, these results support a model that PVH SH2B1 neuron-intrinsic BDNF/TrkB/SH2B1 pathways safeguard the activation and function of the PVH SH2B1 →DRN neurocircuit (Figure 6L).Thus, the PVH SH2B1 →DRN neurocircuit may serve as a new paradigm to investigate brain control of body weight and metabolism.
Ethics Statements: Animal research complied with all relevant ethnic regulations.Animal experiments were conducted following the protocol PRO00011480 approved by the University of Michigan Institutional Animal Care and Use Committee (IACUC).

Figure 7 .
Figure 7. SH2B1 enhances BDNF action in the PVH.A) Males (18 weeks old) were stimulated with BDNF (60 ng per mouse, icv) for 20 min.PVH extracts were immunoblotted with the indicated antibodies.B) Sh2b1 f/f males were bilaterally injected into the PVH with AAV8-hSyn-fDIO-Cre or AAV-GFP while AAV-EF1a-Flpo vector was injected into the DRN.Ten weeks later, mice were stimulated with BDNF (40 ng per mouse, icv) for 20 min.PVH extracts were immunoblotted with the indicated antibodies.C) Sh2b1-Cre males were bilaterally microinjected into the PVH with AAV9-CAG-DIO-SH2B1 or AAV-CAG-mCherry vector, fed HFD for 10 weeks, and stimulated with BDNF (20 ng per mouse, icv).PVH extracts were immunoblotted with the